Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • #16
    Hi again,
    Do not need answer for my question. I found this post where the author answer a similar question. Thank you anyway.

    Discussion of next-gen sequencing related bioinformatics: resources, algorithms, open source efforts, etc


    Carlos

    Comment


    • #17
      quality values for trimmomatic

      I am having trouble understanding the quality value used in trimmomatic. I want to trim bases from 3' and 5' ends that are below Q20. I have Illumina 1.8+ so my quality values are phred64. For the sample script they use 3 as the quality value. This can't be a Qscore of 3 so what is this?

      Comment


      • #18
        Originally posted by shawpa View Post
        I am having trouble understanding the quality value used in trimmomatic. I want to trim bases from 3' and 5' ends that are below Q20. I have Illumina 1.8+ so my quality values are phred64. For the sample script they use 3 as the quality value. This can't be a Qscore of 3 so what is this?

        Please disregard. I was being stupid. My values are in phred33.

        Comment


        • #19
          Trimming 3' end with Trimmomatic

          Hi,

          Would someone please tell me if there is a way to do trimming in Trimmomatic (using sliding window) on 3' end only?

          Thanks

          Comment


          • #20
            Take the example on the web page and only use the 'TRAILING:3 SLIDINGWINDOW:4:15' part of it. I.e., drop the 'ILLUMINACLIP:illuminaClipping.fa:2:40:15 LEADING:3 MINLEN:36' part of the example.

            Comment


            • #21
              OK, Thanks Westerman

              Comment


              • #22
                I have some questions about the folder with the sequences of the adapters:

                1. Where i have to create the folder?
                2. Can anyone give me an example of the format? I'm using Illumina TruSeq adapters.

                Thanks.

                Comment


                • #23
                  Hi all,

                  I started to use Trimmomatic 0.22 three weeks ago and i'm having some difficulties.

                  Until now i was doing the adapter trimming and quality trimming with CLC. In order to check which method is going to give me the better quality of trimmed reads i'm doing again the trimming of my libraries with Trimmomatic and comparing the fastQC reports of both (CLC and Trimmomatic).

                  For some reason Trimmomatic is not doing a very good job with my forward sequences and i'm still having some overrepresented sequences that the fastQC recognize as one of the Illumina indexes.

                  I thought that maybe was because my adapters fasta file was wrong so i was playing around a little bit with it.

                  Now i'm completely lost and exhausted so i need help. Here i attached my 2 adapters fasta files. Because i built my libraries with 2 different kits from Illumina (the Multiplexing Sample Prep Oligo Only kit: with homemade adapters and with Illumina adapters; and the TruSeq kit).

                  And here is my script:

                  for FRW in $(ls *_R1_005.fastq.gz); do
                  for RVS in $(ls *_R2_005.fastq.gz); do
                  bsub -q $queue java -Xincgc -Xms4g -Xmx4g -classpath ../../trimmomatic-0.22/Trimmomatic-0.22/trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticPE -phred33 $FRW $RVS ../trimmomatic_data_trial/$FRW.FRWPE.fq.gz ../trimmomatic_data_trial/$FRW.FRWUnP.fq.gz ../trimmomatic_data_trial/$RVS.RVSPE.fq.gz ../trimmomatic_data_trial/$RVS.RVSUnP.fq.gz ILLUMINACLIP:../../trimmomatic-0.22/TruSeq_adapters.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20


                  THANKS!!!
                  Attached Files

                  Comment


                  • #24
                    Well, your script is confusing. It looks like you'll be comparing multiple forward files to multiple reverse files without any correspondence between the forward and reverse sequences. You then regenerate the same output files over and over again with the last files being generated good for the reverse sequences but not the forward ones.

                    Now if you only have one matching file for your 'ls' the above observation doesn't really matter. But if you have multiple files then ... let's see.

                    Assume the following files:
                    A_R1_005.fastq.gz
                    B_R1_005.fastq.gz
                    C_R2_005.fastq.gz
                    D_R2_005.fastq.gz

                    Which I'll abbreviate as 'A', 'B', 'C', and 'D.

                    A vs C produces files:
                    A.FRWPE.fq.gz A.FRWUnP.fq.gz C.RVSPE.fq.gz C.RVSUnP.fq.gz

                    A vs D produces files: (bad match between R1 and R2)
                    A.FRWPE.fq.gz A.FRWUnP.fq.gz D.RVSPE.fq.gz D.RVSUnP.fq.gz

                    B vs C produces files: (bad match between R1 and R2)
                    B.FRWPE.fq.gz B.FRWUnP.fq.gz C.RVSPE.fq.gz C.RVSUnP.fq.gz

                    B vs D produces files:
                    B.FRWPE.fq.gz B.FRWUnP.fq.gz D.RVSPE.fq.gz D.RVSUnP.fq.gz

                    Everything is confused!

                    Now it is quite possible that your script is working ok (either because of single files or because it was not presented completely) but you may wish to run your Trimmomatic without the loops involved.

                    Comment


                    • #25
                      Hi westerman,

                      Thanks for the answer.

                      So, i only have one forward file (R1) and one reverse file (R2) so i think that even is better to not to use loops the script is not the problem. Before using this script with the loops i was using this:

                      java -Xincgc -Xms4g -Xmx4g -classpath ../../trimmomatic-0.22/Trimmomatic-0.22/trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticPE -phred33 [name_forward_file] [name_reverse_file] ../trimmomatic_data_trial/[name_forward_file]_PE.fq.gz ../trimmomatic_data_trial/[name_forward_file]_UnP.fq.gz ../trimmomatic_data_trial/[name_reverse_file]_PE.fq.gz ../trimmomatic_data_trial/[name_reverse_file]_UnP.fq.gz ILLUMINACLIP:../../trimmomatic-0.22/TruSeq_adapters.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20

                      ... and i was having the same problem with my reverse trimmed sequences. That's why i thought that the problem is in my adapters fasta file.

                      Comment


                      • #26
                        Your adapter fasta files have /2 after the IDs of the index adapters which tells Trimmomatic to only check these sequences against the second (reverse) read. These adapters will show up in the first (forward) read, not the second. Likewise the universal adapter sequence will show up in the second read, not the first. You need to swap the /1 and /2 endings in your adapter FASTA files and you should use the reverse complement of the Universal adapter. You could remove /1 and /2 entirely forcing Trimmomatic to check the adapters against all reads but that is inefficient.

                        Comment


                        • #27
                          Ok, having gotten the possible script problem out of the way, everything else looks syntactically correct. Time to dig into the adapter file or the actual reads that are not being properly trimmed. It is possible that the 2:40:15 parameter to the ILLUMINACLIP is tripping you up. Are you using the simple trim or the, in my experience more rare, palindrome trim? You say:

                          i'm still having some overrepresented sequences that the fastQC recognize as one of the Illumina indexes.
                          What are these sequences?

                          Can you look at a handful of reads reported to have illumina indexes and manually see if they have the index. And, if so, how good is the match?

                          Comment


                          • #28
                            Originally posted by kmcarr View Post
                            Your adapter fasta files have /2 after the IDs of the index adapters which tells Trimmomatic to only check these sequences against the second (reverse) read. ...
                            Ah, good catch! All my adapters are non-strand specific with reverse-complements thus I just skipped over that part of his adapters. It is undoubtedly the correct solution.

                            Comment


                            • #29
                              Thanks a lot!!!

                              Do you recommend me to do an individual FASTA file for each index?

                              Comment


                              • #30
                                All my adapters are in one file. Given the number of adapters I can not see it working otherwise. I undoubtedly over process my sequences by looking at both strands in both directions with all possible adapters -- as kmcarr says, "inefficient" -- but it makes me more comfortable that I am picking up everything.

                                Comment

                                Latest Articles

                                Collapse

                                • seqadmin
                                  Techniques and Challenges in Conservation Genomics
                                  by seqadmin



                                  The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                                  Avian Conservation
                                  Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                                  03-08-2024, 10:41 AM
                                • seqadmin
                                  The Impact of AI in Genomic Medicine
                                  by seqadmin



                                  Artificial intelligence (AI) has evolved from a futuristic vision to a mainstream technology, highlighted by the introduction of tools like OpenAI's ChatGPT and Google's Gemini. In recent years, AI has become increasingly integrated into the field of genomics. This integration has enabled new scientific discoveries while simultaneously raising important ethical questions1. Interviews with two researchers at the center of this intersection provide insightful perspectives into...
                                  02-26-2024, 02:07 PM

                                ad_right_rmr

                                Collapse

                                News

                                Collapse

                                Topics Statistics Last Post
                                Started by seqadmin, 03-14-2024, 06:13 AM
                                0 responses
                                32 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-08-2024, 08:03 AM
                                0 responses
                                71 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-07-2024, 08:13 AM
                                0 responses
                                80 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 03-06-2024, 09:51 AM
                                0 responses
                                68 views
                                0 likes
                                Last Post seqadmin  
                                Working...
                                X